The main aim of the research project is an understanding of the chemical reactivity of flavin coenzymes, and how this reactivity is used and controlled in biological systems. The methods of approach will be the study of specific enzymes representative of the various classes of flavoproteins and studies of model systems. Considerable use will be made of steady state kinetics coupled with rapid reaction kinetics studies, and of the technique of replacing the natural coenzymes of flavoproteins with synthetic flavins, in order to test possible mechanisms, and to probe the nature of the environment immediately around the protein-bound flavin. The latter is particularly important in providing information about the active site region in proteins where the structure is not available from X-ray crystallography. It also offers a powerful way for investigating dynamic aspects of protein structure in the case of enzymes where the crystal structure is known. We are also making extensive use of molecular genetics techniques in the elucidation of chemical reaction mechanisms. We have cloned and expressed the genes for L-lactate monooxygenase, MHPC oxygenase and Old Yellow Enzyme, and are carrying out detailed studies on site-directed mutant forms of these enzymes, and of L-lactate oxidase, D-amino acid oxidase, DT-diaphorase, alkyl hydroperoxide reductase and p-hydroxybenzoate hydroxylase. For all of these enzymes the three dimensional structure is already available, or the structural determination is in progress.